Various flow cytometers and microfluidic systems exist for the purposes of analyzing and separating particles. Each of these instruments has various disadvantages making them less desirable for certain applications. Early flow cytometers were able to count particles in fluid streams and were eventually able to differentiate and count particles having different characteristics, such as different sizes. As new dyes and staining procedures developed, the capacity of flow cytometers to differentiate particle characteristics improved, resulting in a variety of cytometers and techniques for analyzing cells according to their shape, density, size, DNA content, and DNA sequence among other features. The DNA content of a cell can be used to determine a cell cycle, the presence of cancer or, in the case of sperm, can be used to differentiate X-chromosome bearing sperm cells from Y-chromosome bearing sperm cells. These older systems operated with analog acquisition and sort electronics that are unable to differentiate particles within a certain proximity resulting in a large number of particles which could not be analyzed or sorted.
With the introduction of digital signal acquisition and digital signal processing, flow cytometers were able to evaluate increasing numbers of particles per second and make more complex multiple parameter analysis. Ellison et al. (WO 01/28700) describe a multiple digital signal processor configuration for performing operations, such as translations, in parallel with a first digital signal processor. Similarly, Durack et al. (WO 04/088283) describe a sorter with four digital signal processors for acquiring and processing flow cytometer signals to sort particles. Each of Durack et al. and Ellison et al. depend upon a triggering event, after which coincidences are determined and the drop delay is applied to the events in a FIFO manner for sorting. Even in flow cytometers dividing tasks among four digital signal processors capable of operating independently and in parallel, when enough particles are detected in rapid succession it is possible for the system to bottleneck, requiring incoming data representing particle events to be cued. After a certain cue is reached, events are aborted to save processing time. Such bottlenecks and aborts may result in erroneous sort decisions. Each digital signal processor is capable of performing a single task at a time and requires some number of clock cycles to achieve each task. The complex computations required for classifying events and sorting particles at event rates exceeding 40,000 per second can surpass the number of clock cycles four parallel digital signal processors can perform. Therefore, a need exists for a robust methodology and apparatus for precisely tracking parameters surrounding each individual particle and each expected droplet and for executing accurate sort decisions.
A common flow cytometer for sorting is the jet-in-air flow cytometer, such as the one described by Hoffman et al. (WO 01/29538). The jet-in air flow cytometer focuses particles within a fluid stream for analysis and perturbs the fluid stream with an oscillator for separating particles. Perturbing the fluid stream results in the formation of droplets downstream of an inspection zone, at which the particles are interrogated and analyzed. In order to sort particles within the fluid stream, the fluid stream may be charged just before a forming droplet, including the particle of interest, separates at a break off point. The droplet retains the charge and as it passes through an electromagnetic field downstream of the break off point is directed to the desired location.
A precise coordination of the droplet charge signal to the break off of the droplet containing the particle to be sorted is required. Historically, this coordination required empirical, iterative procedures to first determine the drop delay within a period of the drop drive frequency, and then within a fraction of the drop drive frequency. Later, automated systems would appear for calculating the drop delay, such as described in United States Patent Application Publication 2011/0221892 (Neckels et al.). Each of these systems have certain disadvantages associated with previously unrecognized problems associated with particle locations in a droplet effecting the correct window for charging said droplet. This historically static parameter is one of the most important determinations required for performing accurate sort actions. Accordingly, a need exists for an improved method and system for sorting particles, especially for particles which can affect operational parameters of a jet-in-air flow cytometer, such as the drop delay.